PROJECT SUMMARY Diapause, a period of developmental arrest, is an essential survival mechanism in insects for coping with inimical conditions, and it plays a crucial role in the transmission of vector-borne diseases. The objective of this proposal is to understand the molecular mechanisms of diapause in adults of the northern house mosquito, Culex pipiens, the vector of West Nile virus, with the long-term goal of disrupting the mosquito?s biological processes during this vulnerable phase of the life cycle. The Forkhead-box, family O (Foxo) transcription factor has been identified as a key molecular switch that activates the downstream genes which govern diapause traits including extended lifespan, fat accumulation, and enhanced stress resistance in diapausing females of Cx. pipiens. We have three specific goals for this project: 1) Characterize the genes that act downstream of the Foxo pathways to generate the overwintering nutrient storage and consumption. We will identify the downstream components of the Foxo and evaluate their putative roles in diapause energetics using three functional assays: RNA interference (RNAi), solid-state NMR, and ATR FT-IR. 2) Characterize the genes that are linked to the overwintering stress tolerance regulated by the Foxo pathway. We have already identified genes downstream of Foxo that are likely linked to stress tolerance using a ChIP sequencing. We will evaluate their links to organismal resistance using functional assays tailored to the cold and oxidative stresses. 3) Establish a link between the Foxo transcription factor and hypermethylation levels in the histone complex leading to extended lifespan. We will test whether the Foxo transcription factor exerts its effect on histone methylation levels through one or both of the two downstream genes encoding Sfmbt (polycomb group protein) and Ptip (pax transcription activation domain interacting protein). This will be achieved by knocking down the expression of these genes or by injecting chemical inhibitors that target the encoded proteins. We will monitor the impact on the histone methylation levels and on the survival rates of the treated Cx. pipiens. Any alteration in the diapause responses (e.g. diapause prevention, delay in onset or termination, impairment of energy storage or utilization, reduced stress tolerance) could potentially be developed as a tool for disrupting this critical phase of the mosquito life cycle and therefore destroying the mosquito before it can reproduce.